On the role of the purine nucleotide cycle in the isolated working rat heart

The purine nucleotide cycle catalyzes the net reaction: aspartate + GTP + H2O----fumarate + NH3 + GFDP + Pi and leads to regeneration of AMP. In skeletal muscle the cycle's rate of operation increases several fold in response to a corresponding increase in work load, which results in a net increase in citric acid cycle intermediates and in release of ammonia. The same may be expected in heart muscle which, like skeletal muscle, possesses the enzymes of the purine nucleotide cycle. Isolated working rat hearts were therefore perfused for 45 min at low or high work load (0.16 v. 0.42 kg m/min/g dry wt) with glucose (5 mM) as substrate. Release of ammonia into the perfusate as well as the tissue content of citric acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, malate, and oxaloacetate), amino acids (aspartate, glutamate and glutamine), adenine nucleotides and phosphocreatine were measured in freeze-clamped tissue. There was no significant change in the sum of the citric acid cycle intermediates (1.295 v. 1.313 mumol/g dry wt), in aspartate (13.21 v. 14.32 mumol/g dry wt), in glutamate (15.58 v. 15.67 mumol/g dry wt), ATP (19.60 v. 19.17 mumol/g dry wt), ADP (5.00 v. 4.11 mumol/g dry wt), AMP (1.45 v. 1.01 mumol/g dry wt) and phosphocreatine (22.58 v. 25.80 mumol/g dry wt) when low and high work load were compared. Ammonia release was 26 mumol/h/g dry wt and 22 mumol/h/g dry wt at low and high work load respectively. The results suggest that in rat heart the activity of the purine nucleotide cycle does not increase with an increase in work load.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infectious complications in chronic lymphocytic leukaemia

Infections are the major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Predisposition to infection in CLL is mediated through various abnormalities including both the immune defects inherent in the primary disease (impairment in humoral and cellular immunity) and in the further immunosuppression related to the management of CLL. Hypogammaglobulinemia is probably the most important immune defect in terms of risk of severe bacterial infections, its frequency and severity progressing with the duration of the disease. Newer antineoplastic agents such as purine analogues, especially when used in previously treated patients, may be associated with a new spectrum of pathogens (Listeria monocytogenes, Pneumocystis carinii, cytomegalovirus, herpes simplex virus, and mycobacteria) involving T-cell dysfunction. In this review we focus on the clinical characteristics of infections in CLL and on the risk factors involved in the pathogenesis of this complication. Furthermore, we describe the evolving patterns of infections associated with purine analogues and discuss the currently accepted approaches to prophylaxis and treatment.Les complications infectieuses de la leucémie lymphoïde chronique     

高效液相色谱法测定啤酒中四种嘌呤物质

目的:建立啤酒中腺嘌呤、鸟嘌呤、次黄嘌呤、黄嘌呤四种嘌呤组分的高效液相色谱测定方法。方法:啤酒样品脱气,用三氟乙酸和甲酸水解或者用高氯酸水解,调溶液pH值为4.0,上高效液相色谱仪测定分析。结果:啤酒样品中四种嘌呤的含量分别是腺嘌呤25.95 mg/L,鸟嘌呤21.60 mg/L,次黄嘌呤4.35 mg/L,黄嘌呤9.35 mg/L,回收率分别为95.8%、91.3%、90.6%和91.3%。结论:此方法简便可行,检测灵敏度、精密度均满足啤酒检测要求。     

Diltiazem administered before or during myocardial ischemia decreases adenine nucleotide catabolism

Calcium antagonists potentially prevent ATP breakdown, but literature data on this subject are conflicting. We studied the effect of diltiazem on ATP catabolism in rat heart, perfused according to Langendorff. Administration of the drug took place either before or during ischemia, induced by lowering the perfusion pressure. The reduction in flow without diltiazem was 85%. We observed a significantly (P less than 0.001) lower production of purine nucleosides and oxypurines by the ischemic heart, when we gave diltiazem in a dose range of 1 to 100 microM before ischemia. The highest drug concentration reduced purine release by 85%. Due to ischemia, myocardial adenine nucleotide content decreased by 40% (P less than 0.001); this was partially prevented by 5 microM diltiazem (P = 0.4 v. untreated hearts). The drug also effectively reduced purine release, when applied five minutes after the induction of ischemia, but higher concentrations were needed.     

Determination of picogram quantities of oligodeoxynucleotides by stripping voltammetry at mercury modified graphite electrode surfaces

The application of graphite/carbon electrodes modified by a mercury layer for the detection of a picomolar quantity of oligodeoxynucleotides (ODN) is described. The electrochemical characterisation of the Hg-modified graphite/carbon electrode surfaces was performed by cyclic voltammetry in presence of 100 μM Cd(II) ions. An optical microscope was used for the visualization of the surface morphology of the bare and Hg-modified graphite/carbon electrodes. The ODN analysis was performed by the following procedure: (i) the chemical step includes the acidic hydrolysis of the ODN (hODN) sample by 0.5 M HClO₄ for a time t = 30 min at 75 °C. During this step only the purine bases are released from the ODN-chain; (ii) the first electrochemical step includes a potential-controlled reduction of the copper ions (Cu(II)) and accumulation of the purine base residue–Cu(I) complex (hODN–Cu(I) complex) on the Hg-modified graphite/carbon surface; (iii) the second electrochemical step represents the cathodic stripping of the electrochemically accumulated hODN–Cu(I) complex from the electrode surface resulting in a current peak on the voltammogram. The proposed electrochemical method can be used for the determination of different ODN lengths in the analysed solution. The stripping current peak of the electrochemically accumulated hODN–Cu(I) complex increased linearly with the length of ODN used. The detection of acid hydrolysed (A)₈₀ ODN by the above-mentioned procedure is possible down to 250 and 500 pM (related to the monomer) for a 0.4 μm mercury layer modified pyrolytic graphite (0.4 μm Hgm-PGEb) and a glassy carbon electrode (0.4 μm Hgm-GCE), respectively. For the shortest (A)₁₀ ODN a detectable value of 1 nM on the 0.4 μm Hgm-PGEb was observed. Our measurements confirm that Hg-modified graphite/carbon electrodes in the presence of the copper ions are suitable for sensitive electrochemical detection of ODN.     

Leflunomide interferes with pyrimidine nucleotide biosynthesis

Leflunomide is an anti-inflammatory and immunosuppressive agent which blocks proliferation of transformed cells and mitogen stimulated normal lymphocytes but does not block T cell signalling mechanisms at antiproliferative concentrations. These properties are consistent with a mechanism involving interference with nucleotide metabolism. Leflunomide had anti-proliferative activity against all cells tested here. The anti-proliferative activities could be reversed by addition of uridine or cytidine to the cultures although some species and cellular differences were observed. Purine nucleosides had no effect. Measurements of nucleotide pools in a human T cell line and mitogen stimulated rat spleen cells treated with leflunomide showed that leflunomide preferentially reduces pyrimidine nucleotide levels. These results indicate that inhibition of pyrimidine biosynthesis is responsible for the anti-proliferative effects of leflunomide.     

巯基嘌呤甲基转移酶活性和硫鸟嘌呤核苷酸浓度检测在6-MP个体化化疗中的意义

目的　探讨巯基嘌呤甲基转移酶 (TPMT)活性、基因型和硫鸟嘌呤核苷酸 (TGNs)浓度检测对 6 巯基嘌呤 (6 MP)化疗个体化的意义。方法　用放射性核素标记方法测定红细胞TPMT活性 ,用特异引物序列 PCR和限制性片段长度多态性 PCR方法检测低活性者的基因型 ,通过高效液相色谱法(HPLC)检测急性淋巴细胞白血病 (ALL)患儿在服用 6 MP后红细胞内的TGNs浓度。结果　 371名受检者的平均TPMT活性为 (16 .6± 4.5 )U mlpRBCs ,其中≤ 10U mlpRBCs的低活性比例为 8.1%,男性和女性的平均TPMT活性分别为 (16 .8± 5 .0 )U mlpRBCs和 (16 .5± 4.4)U mlpRBCs ,汉族人TPMT活性不存在性别、年龄差异 ,健康志愿者与白血病患儿之间差异亦无显著性 ;30名TPMT活性低下者的DNA中包括TPMT 2型 5例 ,TPMT 3A型 4例 ,TPMT 3B型 6例 ,TPMT 3C型 10例 ,另有 5例未出现上述基因型 ;ALL患儿治疗前红细胞TPMT活性与服用 6 MP 7～ 14d的红细胞内TGNs稳态浓度呈负相关。TGNs浓度与测定后第 14天的白细胞计数呈负相关。结论　服用 6 MP前测定TPMT活性和服药时监测TGNs浓度能够有助于预防抗嘌呤代谢药物的不良反应 ,指导其化疗个体化 ,改善疗效。     

Localisation of the adenosine uptake site in the human brain: a comparison with the distribution of adenosine Al receptors

Using quantitative receptor autoradiography we investigated the distribution of the adenosine uptake site labelled with [³H]NBTI in post-mortem human brain and compared its distribution with that of the A1 adenosine receptor labelled with [³H]CHA. The highest levels of [3H]NBTI binding were found in the cortex and striatum, with moderate levels in the hippocampus, globus pallidus, cerebellum and some midbrain and spinal cord nuclei. The distribution of A1 receptors and this adenosine uptake site differed in the hippocampus where A1 receptors were highest in CAI but the uptake site was low in CA1 and higher in the molecular layer of the dentate gyrus. These results define the anatomical distribution of the high affinity NBTI sensitive adenosine uptake site in the normal human brain.     

Purine- and pyrimidine-stimulated phosphoinositide breakdown and intracellular calcium mobilisation in astrocytes

Phosphoinositide breakdown in cultured cortical astrocytes was assessed by measuring the accumulation of [³H]inositol phosphates (IP's) following incubations with various purines and pyrimidines. Dose-response relationships gave the following order of potency: 2-methylthioadenosine triphosphate (2-MeSATP) > uridine 5′-triphosphate (UTP) > ATP = ADP > inosine 5′triphosphate (ITP). However, 2-MeSATP and UTP were only half as effective as either ATP or ADP in stimulating [³H]IP production. Astrocytes were also challenged with combined additions of maximally effective concentrations of agonists. Responses to ADP plus UTP and 2-MeSATP plus UTP were essentially additive whilst ATP plus UTP evoked a response which was only partially additive. ATP-stimulated [³H]IP accumulation was markedly reduced in the presence of 2-MeSATP suggesting that the latter may be a partial agonist at these receptors. We also examined the ability of ATP and UTP to increase intracellular Ca²⁺ concentrations in these cells. Greater than 90% of all cells tested responded to ATP with a release from internal Ca²⁺ stores but less than half of these responded similarly when challenged with UTP. Our results indicate that astrocytes possess both P_2Y-purinoceptors and a population of receptors which are also coupled to phosphoinositide metabolism and intracellular Ca²⁺ mobilisation but recognise ATP and the pyrimidine nucleotide UTP.     

Second Malignancies and Richter's Syndrome in Patients with Chronic Lymphocytic Leukemia

Second malignancies are frequent complications in patients with chronic lymphocytic leukemia (CLL). Patients with this leukemia may develop large cell lymphoma (LCL) known as Richter's syndrome (RS). RS occurs in CLL patients of about 3% and may develop in a single lymph node or more often in a group of nodes. However, in some patients extranodal localization of aggressive lymphoma in RS has been observed. Besides LCL, Hodgkin's disease, prolymphocytoid leukemia, multiple myeloma and acute lymphoblastic leukemia may also occur as RS variants. The origin of lymphoid cells in RS remains tentative. However, CLL and RS originate from the same clone for some patients, whereas, in other patients cells of aggressive lymphoma do not have the features of the same clone as the CLL cells. The prognosis of RS is poor. Survival in different studies will be usually 2-5 months. The secondary development or coexistence of myeloproliferative disorders or myelodysplastic syndrome and solid tumors have also been rarely documented in CLL patients. It is of great concern that therapy may further increase the risk of a second neoplasm. However, until now, there are no clear evidence that alkylating agents or purine nucleoside analogs may be associated with an increased incidence of second malignancies in patients with CLL. In this review, epidemiology, biology, clinical characteristic and treatment approaches in RS and other secondary neoplasms in patients with CLL are discussed.